弹性蛋白酶基因在大肠杆菌中的高效表达与活性检测.doc

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  • 更新时间:2016-11-03
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摘要:弹性蛋白酶经济价值高,目前国内仅从猪胰脏中提取,由于酶源受到限制,作为治疗用生化药物和其它方面的应用长期供不应求。但是利用微生物开发弹性蛋白酶有着不可估量的前景。

   本实验以实验室原保存的携带弹性蛋白酶前体基因的重组载体pGM-T/ela为模版设计引物,与质粒载体pGM-T连接后得到重组质粒载体,双酶切后得到目的基因片段,与表达载体pET-30a(+)连接后转化进入大肠杆菌,经IPTG诱导后,采用SDS-PAGE检测目的蛋白表达情况。经过弹性蛋白固体培养基打孔实验,观察透明圈方法鉴定其水解蛋白质的活性。

   结果表明,弹性蛋白酶基因在大肠杆菌中高效表达。弹性蛋白基因成功连接到质粒载体pET-30a(+)中,构建成表达载体pET-30a(+)/ela。凝胶电泳结果显示其相对分子质量约为1400bp。将其成功转化进入大肠杆菌。通过SDS-PAGE凝胶电泳,结果显示在31kDa出现一条明显的条带,表示弹性蛋白在大肠杆菌中大量表达,且分子质量约为31kDa。弹性蛋白固体打孔实验出现很明显的水解透明圈,说明此蛋白具有水解弹性蛋白的活性。蛋白表达量占菌体总蛋白的62%。因此,通过大肠杆菌表达系统大量制备具有生物活性的弹性蛋白酶的策略是可行的,研究结果为弥补国内弹性蛋白酶产量短缺提供一种途径。

关键词 弹性蛋白酶;诱导表达;活性检测;重组质粒

 

Abstract:Elastase has a high economic value. Currently we can only get it from the pig pancreas .As an enzyme source is limited, it is a long-term shortage of it in the biochemical drugs and other aspects of the application. Though, it is immeasurable to use the microorganism.

   The elastase gene was cloned from the recombinant vector which harboring the interest gene,and which was then ligated, with the expression vector pET-30a(+).Ligated products were transformed into E.coli.Recombinant protein was analyzed by SDS-PAGE after the IPTG induction.After the the elastin solid medium punch Experiment, we co µld observe transparent circle to dentificate the hydrolyzed protein activity.

   As a result, the elastase gene expressed efficiently in E. Coli, making up 62% of the total protein Elastin gene was successf µlly connected to the plasmid vector pET-30a (+) to construct the expression vector pET-30a(+)/ela. Gel electrophoresis results showed its relative molecular mass was about 1400bp.And it transformed into E.coli successfully.By SDS-PAGE gel electrophoresis, we cloud find that the elastin is expressed. Many hydrolysis transparent rings apeared in the elastin solid medium.It showed that this protein having the activity of the hydrolysis of elastin.So it is possible to largely produce bioactive recombinant forethought the E.coli expression system. These res µlt wo µld provide a method to solve the problem of lack of the elastase.

Keywords  elastase   induced  Detection activity  Recombinant plasmid

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